Using the mouse skin initiation/promotion model the contributions of the PGE2 receptors, EP1, EP2, EP3 and EP4 in papilloma development were investigated. Indomethacin was used to inhibit epidermal PGE2 production and reduced papilloma formation about 60% in initiated/promoted mice. In TPA/indomethacin treated mice the EP2 agonist (butaprost) restored papilloma formation to TPA alone treatment levels, but EP1, EP3 and EP4 agonists were ineffective. In papillomas, the EP2 agonist increased Ras and PKA activation, as well as c-AMP, p-Src, p-EGFR, p-Erk and p-Akt levels. Treatment of papillomas with EGFR (Ag1478) or Src (PP2) inhibitors demonstrated that p-EGFR was downstream of p-Src. AG1478 also inhibited the activation of Ras, Erk and AKT. EP2s role in papilloma development was confirmed by the observations that EP2 -/- mice showed a 60% reduction in papilloma numbers and reduced activation of Ras, Src, AKT and Erk. Immunoprecipitation of p-Src or EP2 indicated the presence of an EP2-B-arrestin1/2-p-Src complex in papillomas of wild type. B-arrestin1/2 can contribute to EP2 internalization as well as facilitate GPCR independent EP2-mediated signaling. Thus, the data indicate that PGE2 acting via EP2 activates signal transduction pathways that can contribute to mouse skin papilloma development in the initiation/promotion model.[unreadable] [unreadable] To further investigate EP2's role in the maintenance of normal skin morphology as well as its contribution to skin tumor formation, the EP2 agonist was applied to naive skin in the absence of TPA or indomethacin. A single treatment of naive skin with butaprost was found to increase epidermal cell replication about 3-fold and to increase PKA and EGFR activation. Furthermore, the PKA and EGFR inhibitors, H89 and AG1478, significantly inhibited butaprost induced epidermal cell replication, and inhibited PKA and EGFR activation. Additionally as was found in papillomas, EP2 formed a complex with Src and B-arrestin. The involvement of B-arrestin in EP2-mediated signaling was confirmed by treating wild type and B-arrestin1-/- fibroblast with butaprost and demonstrating that Src activation was significantly diminished. Thus, the combined data indicate that EP2-mediated signaling contributes to mouse skin tumor formation, and does so by G protein dependent and independent mechanisms.